Cataract disease results when the eye lens becomes opaque and scatters a significant part of the incoming light into the eye. The lens contains very high concentrations of the lens proteins, called crystallins, which are present at concentrations comparable to those found in protein crystals (about 400–600 mg/mL). Chemical modifications of the crystallins, such as oxidation and deamidation, or genetic mutations are known to result in increased light-scattering in vitro, and are implicated in cataract formation in vivo . Here we present the in vitro work on a mutant protein of human γD crystallin (HGD), namely R76S (i.e. Arg 76 to Ser substitution) which is associated with juvenile cataract. Our spectroscopic studies show that the mutant protein has secondary and tertiary structures identical to those of the native protein. Its thermal stability also appears to be unaltered by the mutation. The observed differences between the wild-type and mutant proteins are: (a) the pI of R76S is lower by 0.4 pH units relative to HGD, which is expected since the basic Arg residue is replaced by a neutral Ser, (b) small chemical shift differences in the HSQC spectra of the two proteins; which indicate that there are minor differences in their structure that are localized near the mutation site, and (c) the R76S mutant shows a slightly higher level of Ca²⁺ ion binding than the wild type, although the difference is less than 10%, and is unlikely to lead to lens opacity.

These differences were observed when both proteins were known to be monomeric and in their normally-folded state. However, other differences between the two proteins relate to the aggregation behavior of these proteins: These are: (a) the R76S mutant at pH 7 and 37 °C gives rise to large aggregates (∼200 times larger than HGD) even under reducing conditions (i.e. in the presence of DTT)—conditions under which HGD remains almost totally monomeric, and (b) SDS-PAGE analysis of R76S shows a distinct band corresponding to a tetramer, in addition to the monomer band. The tetramer band is not observed for HGD. When excised and re-electrophoresed in the presence of an excess of DTT, the tetramer band is partially monomerized. Notably however, the tetramer band does not form if the mutant sample is boiled briefly prior to the SDS-PAGE analysis. Heating the protein sample under reducing conditions prior to analysis, leads to monomerization. These data led to our current working hypothesis that minor structural perturbations near the mutation site in R76S render the nearby Cys residue(s) vulnerable to intermolecular S-S crosslinks. Such interactions are likely to be followed by other attractive (for example, hydrophobic) interactions, culminating in the formation of a stable tetrameric species observed in the SDS-PAGE analysis. It is possible that these oligomers are stabilized by SDS as described above; while in its absence, much larger aggregates are observed using dynamic light scattering. These types of large protein aggregates could be responsible for light scattering in vivo. Further studies would be required to substantiate these findings.